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Abstract

Introduction: Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated variant in this region, rs12190287, resides in the 3’ untranslated region of the basic-helix-loop-helix transcription factor, TCF21, and is predicted to alter the seed binding site for miR-224. Allelic imbalance studies demonstrated significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287.

Methods and Results: Reporter assays in human coronary artery smooth muscle cells (HCASMC) showed that the disease-associated C allele is selectively targeted by miR-224, leading to reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor- and platelet derived growth factor signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis in vivo.

Conclusions: Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for CHD at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21, in conjunction with previous studies showing that this variant modulates transcriptional regulation through AP-1, suggests a unique bimodal level of complexity previously unreported for disease-associated variants.